1. Field of the Invention
The present invention relates generally to the field of antibody analysis and generation, such as antibody discovery from immunized animals. More particularly, it concerns novel methods and compositions for identification and/or production of desired antibodies or antigen-binding fragments.
2. Description of Related Art
Over the last 12 years, the development of cancer therapeutic antibodies, such as Herceptin (Trastuzumab, anti-Her2), Rituxan (Rituximab, anti-CD20), Eribitux/Vectibix (Cetuximab/Panitumumab, anti-EGFR), Avastin (anti-VEGF), and others, have saved many tens of thousands of lives world-wide. Antibody therapeutics offer distinct advantages relative to small molecule drugs, namely: (i) better understood mechanisms of action; (ii) higher specificity and fewer-off target effects; (iii) predictable safety and toxicological profiles. Currently, there are more than 200 antibody therapeutics in clinical trials in the U.S., many of them for cancer treatment.
The discovery of monoclonal antibodies is an immensely important aspect in therapeutic antibody development. Additionally, monoclonal antibodies are widely used for numerous diagnostic and analytical purposes. Since the development of the hybridoma technology by Kohler and Milstein 35 years ago (Kohler and Milstein, 1975), a variety of methods for the generation of MAbs have been developed. Such methods include B-cell immortalization by genetic reprogramming via Epstein-Barr virus (Traggiai et al., 2004) or retrovirus-mediated gene transfer (Kwakkenbos et al., 2010), cloning of V genes by single-cell PCR (Wrammert et al., 2008; Meijer et al., 2008), and methods for in vitro discovery via the display and screening of recombinant antibody libraries (Clackson et al., 1991; Feldhaus et al., 2003; Harvey et al., 2004; Schaffitzel et al., 1999; Hosse et al., 2006; Mazor et al., 2007; Zahnd et al., 2007; Kretzschmar and von Ruden, 2002). Both in vitro and in vivo methods for antibody discovery are critically dependent on high-throughput screening to determine antigen specificity. Recently, B-cell analysis has been expedited by microengraving techniques that utilize soft lithography for the high-throughput identification of antigen-specific B cells; however, this is at the cost of considerable technical complexity due to the need for antibody V gene amplification and cell expansion (Jin et al., 2009; Love et al., 2006).
Similarly, the success of in vitro antibody discovery techniques is dependent on screening parameters including the nature of the display platform, antigen concentration, binding avidity during enrichment, multiple rounds of screening (e.g., panning or sorting), and importantly, on the design and diversity of synthetic antibody libraries (Hoogenboom, 2005; Cobaugh et al., 2008; Persson et al., 2006).
Current use of display technologies coupled with library screening systems, such as a phage display where antibodies are isolated by panning, has a number of significant problems. In particular, some antibodies produced by a library may cause the death of the organism expressing them and therefore they simply cannot be detected. There is a particular problem when one is searching for antibodies specific to an antigen from a pathogen that might be homologous to one produced by the host expression system (e.g., E. coli) because, in that instance, important antibodies cannot be expressed. The use of E. coli to express libraries of human antibodies also suffers from the problem of codon usage. Codons used by humans for specific amino acids are frequently not the optimum ones for the same amino acid in E. coli or other host systems. This means that an important antibody might not be expressed (or at least not in sufficient quantities) since the codons in its sequence are highly inefficient in E. coli, resulting in the E. coli being unable to read through and express it in full. Codon optimization of antibody libraries is obviously not an option since the libraries would first have to be sequenced, which defeats the main advantages of using libraries.
There is a pressing need to identify biologically relevant antibodies that exhibit a beneficial effect in controlling diseases. Mammals mount antibody (humoral) immune responses against infectious agents, toxins, or cancer cells. Diseased individuals produce circulating antibodies that recognize the disease agent, and in many cases (e.g., in patients that recover from an infection or in cancer patients in remission) these antibodies play a key role in recovery and therapy. Currently there are no methods available to identify the circulating antibodies in blood and to produce the antibodies that are specific to the disease agent and have a therapeutic effect.
On the other hand, the isolation of monoclonal antibodies from different animal species is of great value for the development of therapeutics and diagnostics. A major limitation of the existing methods for isolation of monoclonal antibodies is that their application is limited to a very small number of species. Different animals have evolved distinct ways of diversifying their antibody repertoire and thus can produce antibodies that recognize distinct epitopes on an antigen or display very high affinity for a particular antigen, compared to mice and humans. For example, it is well known in the art that antibodies from rabbits generally display much higher affinity than those produced from mice.
Current production of monoclonal antibodies from a particular species using hybridoma technology necessitates that B cells are immortalized by fusion to a myeloma from that species. Such myeloma cell lines are difficult and time consuming to develop and therefore exist only for mice, primates, rabbits, and sheep. Alternatively, researchers have attempted to generate interspecies hybridomas, by fusing a mouse myeloma cell line with B cells from an animal for which autologous myeloma cell lines are not available. However, interspecies hybrids are generated with very low efficiency and are unstable, ceasing to produce monoclonal antibodies after a few passages. Thus, at present the production of monoclonal antibodies from the vast majority of animals that have an adaptive immunoglobulin system is a major challenge. Moreover, even for species for which stable B-cell fusions can be generated (rabbits, mice, sheep, and primates) the isolation of monoclonal antibodies using hybridoma technology is a lengthy process requiring 2-6 months after animal sacrifice.
Alternatively monoclonal antibodies can be isolated in vitro from large libraries of the variable (V) chains of the immunoglobulin repertoire from an immunized animal and then screening by a variety of display methods, such as phage display, yeast display, or bacterial display. Once again the utility of these methods is limited to the few species for which extensive information on their immunoglobulin repertoire is available, namely mice, primates, and rabbits. This is because the cloning of the immunoglobulin repertoire requires the availability of sets of oligonucleotide primers capable of amplifying the majority, preferably all, of the immunoglobulin variable regions that are generated in that animal via somatic recombination mechanisms. This in turn requires extensive information on the sequences of immunoglobulins expressed in a particular species and it is not available for the vast majority of animals that have an antibody-encoding, humoral immune system. Additionally, it is not known whether the antibodies isolated by combinatorial library screening correspond to those that have been expanded by the immune system and produced in large amounts in animals.
All of these techniques are somewhat complex, inconvenient, and time consuming. Therefore, there remains a need to develop a more efficient and accurate method for identifying antigen-specific antibodies or monoclonal antibodies directly from a patient or any animal.